A fundamental problem in nucleic acid analysis is sample preparation. The sample to be investigated usually comprises cells or tissue with interfering, partially insoluble constituents (known as debris) which can interfere with the subsequent isolation and analysis. Such insoluble constituents occur particularly in the case of nucleic acid isolation from stool/feces, blood, warts, calcified structures (bones), or else heavily necrotic tissue samples. However, debris can, in the broadest sense, also include soluble components, for example released hemoglobin from erythrocytes which is present in a great excess and will be removed during the isolation of the nucleic acids.
Isolation of nucleic acids from samples such as cells, tissues, plants, bacteria, viral particles, blood, serum, or plasma, is a critical step for downstream genetic analysis. Conventionally, liquid phase extraction techniques, such as phenol/chloroform precipitation, are widely used. Although these approaches yield nucleic acids of high quality, they are laborious, time-consuming and highly operator-dependent. Solid phase extraction techniques are a popular alternative. They are often the methods of choice when processing large numbers of samples. Commonly used solid-phase substrates include silica spin columns and silica magnetic particles that provide large surface areas for nucleic acid binding. However these porous matrices and micro/nano particles induce DNA shearing as a result of flow and particle mixing, leading to decreased DNA integrity.
Molecular analysis of Formalin Fixed Paraffin Embedded (FFPE) samples represents another area where advances in sample preparation are needed. Despite the growing need for and the demonstrated potential advantages of molecular biomarkers, it has proven difficult to routinely employ them in the diagnosis and management of patients. One reason for this failure has been the logistical challenges of obtaining, rapidly processing, storing, and transporting quick-frozen tissue samples in clinical settings. Standard hospital tissue processing involves fixation in formaldehyde, followed by embedding in paraffin blocks, then by subsequent sectioning and staining of these blocks to generate FFPE samples.
If these FFPE samples could be harnessed for molecular analysis, the potential for revolutionizing current medical practice exists. FFPE blocks obtained in hospital pathology departments could then be routinely assayed using the newer molecular methods, in addition to standard morphological and histological analysis. Moreover, since FFPE samples are usually stored for many years by hospital pathology departments, retrospective molecular evaluations could also be performed, empowering researchers to conduct molecular epidemiologic studies on large cohorts with known clinical outcomes. New technologies are needed such that molecular pathologic assays could be devised or adapted to work on these FFPE samples.
However, as FFPE preservation was originally designed to stabilize morphological and histological features rather than preserve molecular information, the DNA/RNA contained within are often fragmented due to the FFPE preservation process, due to oxidation, and due to poor storage conditions (i.e. long-term archival at room temperature). In addition, FFPE tissues contain contaminating formalin and paraffin wax as well as heavily cross-linked DNA/RNA that can inhibit downstream assays.
As such, there exists an unmet need to develop novel separation materials and methods which allow for easier isolation and purification of nucleic acids from a clinical sample, including FFPE samples.